Despite recent improvements in standard care, patients with many malignant lesions such as pancreatic cancer still have poor prognoses. Oncolytic virotherapy is a promising alternative treatment that may provide therapeutic benefit to these patients. Cancer virotherapy functions by converting the natural cell killing ability of a benign virus to selectively kill cancer cells. Significant progress has been made in recent years in developing this therapeutic modality. Recent data from a phase III clinical trial using an HSV-1-based oncolytic virus, T-VEC, show an increase of 4.4 months in overall survival when compared with GM-CSF treatment alone (p=0.051). While these clinical data are clearly encouraging, they also indicate that the current cancer virotherapy needs further improvement before it may eventually gain the FDA approval. We have developed an HSV-2-based oncolytic virus, the first of this kind. Designated FusOn-H2, it can selectively replicate in tumor cells bearing an activated Ras signaling pathway. When evaluated in vivo, it can effectively shrink or even eradicate many malignant tumors, including pancreatic cancer, which harbors a high frequency (over 90%) of Ras mutation. In this proposal, we will explore a series of novel strategies to potentiate FusOn-H2 in several key steps of virotherapy, including enhancing delivery, reducing therapy resistance and adaptation to tumor microenvironment, primarily using pancreatic cancer as a tumor model. Our working hypotheses are: 1) Arming FusOn-H2 with a don't eat me signal will allow the virus to persist in the blood, and incorporation of echistatin which has a strong binding affinity to av3 integrin on endothelial cells of tumor neovasculature, into the virus will enable it to be actively delivered to tumor tissues by the systemic route. 2) Te dynamic interactions between the Ras-ERK and PI3K-Akt-mTOR pathways dictate the permissiveness of pancreatic cancer cells to FusOn-H2. Small molecule drugs will be explored to manipulate these interactions to increase the permissiveness of pancreatic cancer cells to FusOn-H2 virotherapy. 3) The arginine-deprived tumor microenvironment, frequently detected in many tumors and caused by overexpression of arginase, presents as an obstacle for oncolytic HSV virotherapy, as early studies have shown that arginase can efficiently inhibit HSV replication. Adapting FusOn-H2 to arginine-deprivation can overcome this obstacle. We have designed three specific aims to test these major hypotheses. The ultimate goal of this proposal is to translate this HSV-2- based oncolytic virus into clinical application, primarily for the treatment of pancreatic cancer - a malignant disease that has an extremely poor prognosis. The novel strategies that we propose to incorporate into FusOn- H2 are expected to significantly enhance the overall therapeutic efficacy of this virotherapy, thus increasing the likelihood of its clinical success. Moreover, many of these strategies may be applicable to other oncolytic viruses for other tumor treatment, thus benefiting the entire field of cancer virotherapy.